This study showed that the V1-V5 regions of HIV-C envelope underwent significant changes in their biological characteristics throughout disease progression. Envelopes from early infection were more positively charged, neutralization sensitive and had faster ex-vivo replication and enhanced fusion capability. During early AIDS, the envelopes became more negatively charged with diminished functional capabilities ranging from ex-vivo replication to incorporation into virions. However, these envelopes did have significantly improved CD4 binding ability and were more neutralization resistant. Interestingly, the envelopes showed multiple reversions in their biological properties during advanced AIDS. For example, their charges, neutralization sensitivity and CD4 binding efficiency had all reverted to pre-AIDS levels. However, other functions remained relatively poor.
Our results are surprising given the current belief that viral fitness increases over time due to envelope evolution 
. It is possible that the previous studies were unable to capture the early infection in adult HIV-1 patients and used PBMC co-cultured viral quasi-species for biological analysis. Our study represents envelope isolates throughout the disease spectrum, focusing on V1-V5 region changes. At the early infection of 6 mpi, monkey infant RPn-8 had an unambiguously higher CD4+
T-cell counts of >2000 cells/ul, which was age-appropriate, and high viral loads compared to reports of other investigators, reflecting acute infection 
. Moreover, having an isogenic backbone allowed us to focus on the V1-V5 region without the influences from other viral genes. Our use of PBMC for ex-vivo
replication also better represents the in-vivo
scenario than the cell lines used in previous study 
. Alternatively, the different maturation state of the immune system between adults and infants during the initial infection might affect the evolutionary pressure placed on the virus.
Our study suggests that during early infection, the host immune system is intact and exerts selective pressures on the viruses, as the relatively early plasma can easily neutralized the early envelopes. To counter this host constraint, the envelope evolved compensatory mutations that resulted in more efficient fusion and faster replication kinetics, increasing the likelihood for the virus to infect new cells before being neutralized by the immune system. This possibility that early envelopes might have better fitness was mathematically predicted previously and observed recently in HIV-1 patients 
. Although the exact mechanism of this outcome is still controversial, the “Red Queen” hypothesis was proposed as a possible explanation, where beneficiary mutations evolved in responses to immune selection resulting in increased fitness of the early envelopes 
During early AIDS, some immune pressure persisted as evident by the presence of neutralization-resistant envelopes and was also reported by others 
. Importantly, the appearance of neutralization-resistant phenotypes coincided with a significant drop in the net charges of V1-V5 region. This strongly suggests that variations in net charges could be an escape mechanism employed by the envelope to evade humoral responses beside its glycan shield 
. The high CD4 binding ability of the envelopes at this stage could be another compensatory mutation for more efficient infection in an environment with limited availability of CD4+
Another unexpected finding from our study is that viral fitness linked to the V1-V5 region, as defined by the ex-vivo
replication of isogenic viruses differing only in their V1-V5 regions, did not improve despite reversion of several envelope properties during advanced AIDS. Importantly, the gp120 content on cell surface and on virion gradually decreased as disease progressed. Additionally, with the exception of the CD4 binding site mutant 6 month #5, decreased levels of gp120 on virion correlated with the decline in fusion ability from early infection to advanced AIDS (Spearman r
0.0214) (). These changes could be evasive responses toward immune surveillance and ultimately affected the neutralization sensitivity as suggested earlier 
. The severe depletion of CD4+
T cells and absence of immune selection might facilitate the accumulation of deleterious mutations, possibly yielding envelopes with lower fitness reminiscent to the effect of “Muller’s ratchet” hypothesis 
. The lack of immune surveillance during the advanced AIDS might allow accumulation of viruses with lower fitness envelopes, resulting in the increase of plasma viral load.
Our study is based on retrospectively collected specimens from an animal that developed AIDS and no other specimens or animals were available for further analysis, the changes we observed could be only confined to this animal. However, the lessons we learned from this study cannot be underestimated, as this is the first study of a SHIV-C-infected macaque that progressed to AIDS. To our knowledge, this is also the first study that demonstrated a decrease of gp120 content on virion with disease progression. Although gp120 shedding might reduce the gp120 content on virion, it was unlikely in our case as suggested by other investigators 
. By focusing on the V1-V5 region, we cannot eliminate the role of other viral genes or envelope regions, such as the signal peptide and gp41, in the overall viral fitness 
. However, our data strongly suggest that V1-V5 region plays a major role in disease progression 
. Also, the presence of mixed population at any time points due to reactivation of latent viral reservoirs cannot be excluded.
Our findings underscore the dynamic interaction between host immune selection and HIV-1 envelope evolution. Our emphasis on the V1-V5 region had shed light on the evolution of its unique properties during disease progression. Moreover, variations in the envelope CD4 binding and fusion ability might suggest some corresponding structural changes with disease progression, which could have substantial implications for the development of vaccines and small molecule inhibitors.